FIGURE 3 from Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects

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posted on 2024-05-22, 14:20 authored by Alexandria P. Eiken, Audrey L. Smith, Sydney A. Skupa, Elizabeth Schmitz, Sandeep Rana, Sarbjit Singh, Siddhartha Kumar, Jayapal Reddy Mallareddy, Aguirre A de Cubas, Akshay Krishna, Achyuth Kalluchi, M. Jordan Rowley, Christopher R. D'Angelo, Matthew A. Lunning, R. Gregory Bociek, Julie M. Vose, Amarnath Natarajan, Dalia El-Gamal

SpiD3 induces the unfolded protein response and modulates CLL survival factors independent of TME stimuli. A, HG-3 and OSU-CLL cells were treated for 4 hours with SpiD3 (5, 10 µmol/L), thapsigargin (Thaps; 10 µmol/L), or equivalent DMSO vehicle (VEH) and then incubated with TPE-NMI dye to probe for unfolded proteins. Data are represented as fold change in TPE-NMI MFI compared with VEH (n = 3–5 independent experiments/cell line). B, Representative immunoblot analyses of IRE1α, XBP1, PERK, ATF4, CHOP, p-eIF2α (Ser51), and total eIF2α in HG-3 and OSU-CLL cells treated with VEH, SpiD3 (0.5–2 µmol/L), or Thaps (2 µmol/L) for 4 hours (n = 4–5 independent experiments/cell line). β-ACTIN served as the loading control. Blue arrow: spliced XBP1, green arrow: PERK shift. C, HG-3 cells were treated with VEH (24 hours), SpiD3 (0.5–2 µmol/L; 24 hours), or cycloheximide (CHX; 50 µg/mL; 30 minutes) and then incubated with OPP for 30 minutes. Data are represented as fold change in OPP MFI compared with VEH (n = 3–4 independent experiments). D, Representative immunoblot analyses of PDCD4, eIF4A1, p-4E-BP1 (Ser65), total 4E-BP1, eIF4E, and eIF4G1 levels in HG-3 and OSU-CLL cells treated with VEH or SpiD3 (0.5–2 µmol/L) for 4 hours (n = 4 independent experiments/cell line). β-ACTIN served as the loading control. Black arrows indicate the three isoforms of 4E-BP1. E, OSU-CLL cells were incubated with the alkyne-tagged analog 19 (10 µmol/L) for 2 hours. Cell lysates were clicked with TAMRA-biotin and biotin-tagged-19-bound proteins were isolated using streptavidin agarose beads, trypsinized, and then evaluated via mass spectrometry. Top: Pathway enrichment (EnrichR) analysis of similar proteins found in at least two of the three biological replicates. Bottom: Representative immunoblot analysis of biotin-alkyne-tagged proteins, with their corresponding input lysates for IKKα, IKKβ, p65, RELB, BTK, and GAPDH. The blue arrow indicates IKKα and IKKβ. Immunoblot analysis of the indicated proteins in whole-cell lysates of HG-3 and OSU-CLL cells treated with SpiD3 (1, 2 µmol/L) for 4 hours and cocurrently stimulated with α-IgM (10 µg/mL; F) or rhBAFF ligand (50 ng/mL; G). α-TUBULIN served as the loading control (n = 4 independent experiments/cell line). Asterisks denote significance versus VEH: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Funding

HHS | NIH | National Cancer Institute (NCI)

HHS | NIH | National Institute of General Medical Sciences (NIGMS)

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ARTICLE ABSTRACT

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NFκB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacologic agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NFκB signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its antileukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NFκB signaling and the UPR, culminating in profound antitumor properties independent of TME stimuli. SpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NFκB signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NFκB pathway and UPR) highlighting its use in drug-resistant CLL.

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Keywords

Hematological Cancers Drug Resistance Preclinical Models In vitro models of cancer Cellular Stress Responses Small Molecule Agents Kinase inhibitors

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